Abstract The transition from replication to non-replication underlies much of Mycobacterium tuberculosis (Mtb) pathogenesis, as non- or slowly replicating Mtb are responsible for persistence and latency and for the long treatment times required to clear Mtb infection. Defining the molecular programs that drive the transition to a state of slow or no replication is central for understanding Mtb pathogenesis and for discovering new drugs that can shorten treatment times. However, there are currently no effective tools to directly identify druggable enzyme activity in the non-replicating state. While previous studies have measured genome-wide transcript or protein abundance in non-replicating cells, these measurements are only approximations of biological activity. A powerful approach to directly measure global enzymatic activity is activity-based protein profiling (ABPP). ABPP directly measures biochemical activity of entire families of enzymes by recognizing a shared catalytic mechanism. This approach is particularly suited for the detection of serine hydrolase (SH) activity, a large enzyme family central to all aspects of metabolism that is extensively regulated by posttranslational modifications. The central role of SHs in many metabolic pathways makes SHs unique reporters for a broad cross-section of Mtb metabolism, and combined with their proven druggability, also tractable drug targets. We developed a chemical proteomics approach to directly measure global SH activity and to define the activity changes between replicating and non-replicating Mtb. We show that SHs are extensively regulated depending on Mtb's replication state. We defined three groups of coordinately regulated SHs in the Mtb life cycle, each indicating a different role in inducing or maintaining the non-replicating state. Here, we will test the hypothesis that a small group of coordinately regulated SHs active ONLY during non-replication are regulators of and potential drug targets in non-replicating Mtb.